Effect of source and level of vitamin D on immune function in growing broilers

Journal of Applied Poultry Research

Volumn 13, Issue 2, 2004, Pages 263-273

  Fritts, C A ^a   Erf, G F ^a   Bersi, T K ^a   Waldroup, P W ^a  

^a UNIVERSITY OF ARKANSAS   (United States)

Author keywords

25 hydroxycholecalciferol; Broiler; Cholecalciferol; Immune function

Indexed keywords

AVES; GALLUS GALLUS; MAMMALIA;

EID: 3142689022     PISSN: 10566171     EISSN: None     Source Type: Journal    
DOI: 10.1093/japr/13.2.263     Document Type: Article

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47. 6 macrophages/mL) from each bird were cultured in 24-well culture plates (4 wells per bird), with or without 10 μg/mL LPS [35] in 1 mL LM Hahn mediumto determine the effects of diet on the production of NO, (measured as nitrite) and macrophage cytotoxicity (measured as percentage killing of RP9 tumor cells). After 24 h, the culture supernatant from each well was collected.
48. Briefly, 100 μL AEC culture supernatant fluid from each sample were added to 100 μL of Griess reagent (1 part 0.1% naphthylenediamine dihydrochloride to 1 part of 1% sulfanilamide in 5% phosphoric acid) in 96-well plates. After 10 min of incubation at room temperature, color intensity indicative of nitrite was quantified by reading the plates at 540 nm absorbance in a microplate reader [37].
49. 2. After 18 h, 50 μL MTT acid (2 parts 2-propanol, 1 part PBS, and 0.0132 parts hydrochloride) were added to each of the wells to identify live RP9 cells and incubated for another 4 h. After the 4 h incubation, plates were centrifuged at 450 x g at 4°C for 4 min. All liquid was then removed from the wells using a multichannel pipettor, being careful not to disturb the crystals. The MTT acid was added again at 150 μL to each well and mixed until all crystals were dissolved. Plates were read at 540 nm in a microplate reader.
50. Blood smears were prepared by placing a small drop (3 to 5 μL) of blood on a clean microscope slide and using another clean slide to push the blood across the slide to spread the blood evenly. Wright stain (adapted for avian blood as described by Lucas and Jamroz [44]) was used to stain leukocytes.